HMGN polypeptides as immune enhancers and HMGN antagonists as immune suppressants

ABSTRACT

A method of enhancing an antigen-specific immune response in a host comprising administering to the host an HMGN polypeptide comprising at least one of HMGN1, HMGN3a, HMGN3b, HMGN4, Nsbp1, or a functional fragment thereof, in an amount effective to enhance an antigen-specific immune response; as well as a pharmaceutical composition comprising an HMGN polypeptide comprising at least one of HMGN1, HMGN3a, HMGN3b, HMGN4, Nsbp1, or a functional fragment thereof, and an antigen, or nucleic acids encoding such molecules; and related methods and compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 12/509,088, filed Jul. 24, 2009, which claims the benefit ofU.S. Provisional Patent Application No. 61/083,781, filed Jul. 25, 2008,each of which is incorporated by reference in its entirety herein.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY FILED

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 10,598 Byte ASCII (Text) file named“710652ST25.TXT,” dated Jun. 25, 2012.

BACKGROUND OF THE INVENTION

HMGN polypeptides belong to the high mobility group (HMG) family ofchromosomal binding peptides. HMGN polypeptides typically functioninside the cell nucleus to bind to DNA and nucleosomes and regulate thetranscription of various genes. HMGN polypeptides also can be releasedby peripheral blood mononuclear cells.

A patient's immune response often plays an important role in theprogression of disease and the effectiveness of medical treatments fordisease. Two types of immune responses can occur in a patient: a Th-1pro-inflammatory type and a Th-2 anti-inflammatory type. The Th-1(cell-mediated) type of immune response activates T-cells andmacrophages, while the Th-2 (antibody-mediated) type of immune responseactivates B-cells.

Often, due to the effects of a disease or even the treatments for adisease, a patient's immune response is diminished or the immuneresponse is disadvantageously shifted away from a Th-1 pro-inflammatorytype response and towards a Th-2 anti-inflammatory type response. Thisdiminished or Th-2 polarized immune response is thought to beresponsible, at least in part, for the more rapid progression of diseaseand reduction in the effectiveness of some treatments. This is thoughtto be true, for example, in cancer patients. In the context of otherdiseases, a heightened or Th-1 polarized immune response can bedisadvantageous and at least partly responsible for the progression ofthe disease or reduction in the effectiveness of treatment. Thus, thereis a need in the art for methods of modulating an immune response.

BRIEF SUMMARY OF THE INVENTION

The invention provides a method of enhancing an antigen-specific immuneresponse in a host comprising administering to the host an HMGNpolypeptide comprising HMGN1 (SEQ ID NO: 1), HMGN3a (SEQ ID NO: 2),HMGN3b (SEQ ID NO: 3), HMGN4 (SEQ ID NO: 4), Nsbp1 (SEQ ID NO: 5), or afunctional fragment thereof, in an amount effective to enhance anantigen-specific immune response.

In another aspect, the invention provides a method of enhancing theactivation or recruitment of dendritic cells in a host comprisingadministering to the host an HMGN polypeptide comprising HMGN1 (SEQ IDNO: 1), HMGN3a (SEQ ID NO: 2), HMGN3b (SEQ ID NO: 3), HMGN4 (SEQ ID NO:4), Nsbp1 (SEQ ID NO: 5), or a functional fragment thereof, in an amounteffective to enhance the activation or recruitment of dendritic cells ina host.

The invention also provides a method of shifting the Th-1/Th-2 balanceof an immune response of a host towards a Th-1 type immune responsecomprising administering to the host an HMGN polypeptide comprisingHMGN1 (SEQ ID NO: 1), HMGN3a (SEQ ID NO: 2), HMGN3b (SEQ ID NO: 3),HMGN4 (SEQ ID NO: 4), Nsbp1 (SEQ ID NO: 5), or a functional fragmentthereof, in an amount effective to shift the Th-1/Th-2 balance of animmune response of a host towards a Th-1 type immune response.

The invention further provides a pharmaceutical composition. Accordingto one aspect of the invention, the pharmaceutical composition comprises(a) an HMGN polypeptide comprising HMGN1 (SEQ ID NO: 1), HMGN3a (SEQ IDNO: 2), HMGN3b (SEQ ID NO: 3), HMGN4 (SEQ ID NO: 4), Nsbp1 (SEQ ID NO:5), or a functional fragment thereof, and (b) an antigen, such as atumor antigen. According to another aspect of the invention, thepharmaceutical composition comprises (a) a nucleic acid that encodes anHMGN polypeptide comprising HMGN1 (SEQ ID NO: 1), HMGN3a (SEQ ID NO: 2),HMGN3b (SEQ ID NO: 3), HMGN4 (SEQ ID NO: 4), Nsbp1 (SEQ ID NO: 5), or afunctional fragment thereof, and (b) a nucleic acid that encodes a tumorantigen.

In another aspect, the invention provides a method of suppressing animmune response in a host comprising administering to the host an HMGNpolypeptide antagonist, wherein the HMGN polypeptide comprises HMGN1(SEQ ID NO: 1), HMGN3a (SEQ ID NO: 2), HMGN3b (SEQ ID NO: 3), HMGN4 (SEQID NO: 4), or Nsbp1 (SEQ ID NO: 5), in an amount effective to suppressthe immune response.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1A illustrates by way of several graphs the concentration ofvarious cytokines (IL-6, IL-8, IL-12p70, and TNFα) in dendritic cellcultures treated with different concentrations of HMGN1 (SEQ ID NO: 1)or HMGN2. FIG. 1B illustrates by way of several graphs the concentrationof various cytokines in control dendritic cell cultures (no treatment)and dendritic cell cultures treated with 1 μg/ml of HMGN1 (SEQ ID NO: 1)or HMGN2 as a function of time. In FIGS. 1A and 1B, data from controlcultures is referenced by -◯-, cultures treated with HMGN1 by -●-, andcultures treated with HMGN2 by -▴-.

FIG. 2 presents flow cytometry histograms showing the proportion ofcells (by number) expressing surface costimulatory molecules (CD80,CD83, and CD 86) and surface major histocompatibility complex (MHC)molecules (HLA-ABC and HLA-DR) in control dendritic cell cultures (notreatment or liposaccharide (LPS)-treated) and dendritic cell culturestreated with HMGN1 (SEQ ID NO: 1) or HMGN2. Surface expression of CD11cwas measured as an additional control. Surface molecule expression isillustrated as a function of fluorescence intensity. The open-areacurves represent staining with isotype-matched control antibody, and theshaded area curves represent staining with antibodies to the varioussurface molecules.

FIG. 3 is a graph of T-cell proliferation (as represented by tritiatedthymidine incorporation) plotted against the ratio of dendritic cells toT-cells (DC:T) in dendritic cell/mixed lymphocyte reaction cultures.Treated cultures contained dendritic cells (DCs) exposed to 1 μg/mlHMGN1 (SEQ ID NO: 1) (-▴-), HMGN2 (-▾-), or LPS (-♦-). Untreated culture(contained sham-treated DCs (-●-)) and mixed lymphocyte reaction culturewithout dendritic cells (-□-) served as controls.

FIG. 4 is a graph of dendritic cell migration (number of cells per highpower field (No./HPF)) towards various cytokines (CCL5, CCL21, andCXCL12) in cultures treated with 1 μg/ml HMGN1 (SEQ ID NO: 1) (diagonalstriped bars) or LPS (black bars), and in untreated culture (containedsham-treated DCs (white bars)). Cell migration without any cytokine wasmeasured as an additional control.

FIG. 5 is a graph of the migration index of subpopulations of DC cells(CD11c+; CD11c+/CD11b+; CD11c+/B220+; and CD11c+/CD11b+/B220) in theperitoneal cavity of mice treated with HMGN1 (SEQ ID NO: 1) (black bars)and control mice treated only with phosphate buffer (PBS) (white bars).

FIG. 6A is a graph of cell proliferation (as represented by tritiatedthymidine incorporation) plotted against ovalbumin (OVA) concentrationin cultures of OVA-specific splenocytes harvested from mice treated withOVA alone (-◯-) or OVA mixed with alum (-♦-) or HMGN1 (SEQ ID NO: 1)(-▴-). FIG. 6B presents graphs of the concentration of various cytokinesin the same cultures.

FIG. 7A presents graphs of the concentration of various cytokinesproduced in response to in vitro stimulation with OVA by splenocytesfrom HMGN1 KO (HMGN1−/−) or wild-type (HMGN1+/+) mice immunized with OVAin the presence of alum. FIG. 7B presents graphs of the concentration ofvarious cytokines produced in response to in vitro stimulation with OVAby splenocytes from HMGN1 KO (HMGN1−/−) or wild-type (HMGN1+/+) miceimmunized with OVA in the presence of LPS.

FIG. 8A is a graph of the number of primary anti-anthrax vaccineadsorbed (AVA) antibodies produced in mice immunized with AVA alone orin the presence of 1 μg or 5 μg of HMGN1 (SEQ ID NO: 1). FIG. 8B is agraph of the number of secondary anti-AVA antibodies produced in miceimmunized with AVA alone or in the presence of 1 μg or 5 μg of HMGN1(SEQ ID NO: 1).

FIGS. 9A and 9B are graphs of the amounts of various cytokines (pg/ml)produced by dendritic cells treated with 0, 0.1, or 1 μg/ml HMGN1 (SEQID NO: 1).

FIG. 10A is a graph of the concentration of various cytokines producedby HMGN1 KO (HMGN1−/−) (white bars) or wild-type (HMGN1+/+) (black bars)mice 24 hours after injection with OVA alone or OVA in the presence ofalum or LPS. FIG. 10B is a graph of the concentration of variouscytokines produced by HMGN1 KO (HMGN1−/−) (white bars) or wild-type(HMGN1+/+) (black bars) mice 96 hours after injection with OVA alone orOVA in the presence of alum or LPS.

FIG. 11 presents a Western blot of untreated or HMGN1 (SEQ ID NO:1)-treated (20 or 60 minutes) DC lysates probed with anti-I-κBα,anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH),anti-phosphorylated p44/42 mitogen-activated protein kinases (MAPKs),anti-p44/42 MAPKs, anti-phosphorylated p38 MAPK, anti-p38 MAPK,anti-phosphorylated c-Jun N-terminal kinase (JNK)MAPK, or anti-JNK MAPKantibodies.

DETAILED DESCRIPTION OF THE INVENTION

HMGN polypeptides are members of the high mobility group (HMG) family ofchromosomal binding polypeptides. The HMG family is subdivided intothree subfamilies, each of which has a characteristic functionalsequence motif: HMGB (HMG-box motif), HMGN (nucleosomal binding domain),and HMGA (AT-hook motif). HMGN polypeptides include HMGN1 (formerlyknown as HMG14) (SEQ ID NO: 1), HMGN2, HMGN3a (SEQ ID NO: 2), HMGN3b(SEQ ID NO: 3), HMGN4 (SEQ ID NO: 4), and Nsbp1 (NBD-45) (SEQ ID NO: 5).

It has been discovered that certain HMGN polypeptides can enhance anantigen-specific immune response. Thus, the invention provides methodsof using HMGN polypeptides and functional fragments thereof to enhancean immune response in a host.

In one embodiment, the invention provides a method of enhancing anantigen-specific immune response in a host comprising administering tothe host an HMGN polypeptide comprising HMGN1 (SEQ ID NO: 1), HMGN3a(SEQ ID NO: 2), HMGN3b (SEQ ID NO: 3), HMGN4 (SEQ ID NO: 4), Nsbp1(NBD-45) (SEQ ID NO: 5), or a functional fragment thereof (hereincollectively referred to as “an HMGN polypeptide”), in an amounteffective to enhance an antigen-specific immune response.

An antigen specific-immune response can be characterized by theproduction of lymphocytes that are capable of recognizing anddifferentiating the antigen from other antigens and mediating thedestruction of the antigen. An antigen-specific immune response also canbe characterized by the production, maturation, activation, orrecruitment of antigen presenting cells.

An antigen-specific immune response is enhanced in accordance with theinvention if the immune response to a given antigen is greater,quantitatively or qualitatively, after administration of an HMGNpolypeptide as compared to the immune response in the absence of theadministration of an HMGN polypeptide. A quantitative increase in animmune response encompasses an increase in the magnitude or degree ofthe response. The magnitude or degree of an immune response can bemeasured on the basis of any number of known parameters, such as anincrease in the level of antigen-specific cytokine production (cytokineconcentration), an increase in the number of lymphocytes activated(e.g., proliferation of antigen-specific lymphocytes) or recruited,and/or an increase in the production of antigen-specific antibodies(antibody concentration), etc. A qualitative increase in an immuneresponse encompasses any change in the nature of the immune responsethat renders it more effective at combating a given antigen or disease.By way of illustration, an antigen-specific immune response typicallyincludes two types of immune responses that occur simultaneously andexist in a relative balance: a Th-1 type response and a Th-2 typeresponse. For the purposes of this invention, the quality of an immuneresponse is considered enhanced in quality if the relative balance ofthe immune response is shifted towards the Th-1 type immune response andaway from the Th-2 type immune response. Methods of distinguishing andmeasuring the relative balance of an immune response are known in theart. For example, measuring the types and levels of cytokines producedcan distinguish and measure the relative balance of an immune response.A shift towards the Th-1 type response (e.g., after administration of anHMGN polypeptide) may be characterized by an increase in IFNγ and noincrease or a reduced increase in IL-4, IL-5, and/or IL-13 (e.g., ascompared to the levels of these cytokines before administration of anHMGN polypeptide). In other words, a shift towards a Th-1 type responsecan be characterized by an increase in the proportion of IFNγ relativeto IL-4, IL-5, and/or IL-13. Conversely, a shift towards the Th-2 typeresponse may be characterized by an increase in IL-4, IL-5, and/or IL-13and no increase or a reduced increase in IFNγ (e.g., an increase in theproportion of IL-4, IL-5, and/or IL-13 relative to IFNγ). Anotherexemplary method may include measuring the subtypes of antigen-specificIgG antibodies produced during an immune response. A higher level(concentration) of IgG2a antibodies versus IgG1 antibodies suggests aTh1-type immune response. Conversely, a higher level (concentration) ofIgG1 antibodies versus IgG2a antibodies suggests a Th2-type immuneresponse. Qualitative and quantitative enhancements in an immuneresponse can occur simultaneously, and are not mutually exclusive.

Preferably, the antigen-specific immune response is enhanced by shiftingthe Th-1/Th-2 balance of an immune response towards a Th-1 type responseand away from a Th-2 type response, i.e., by enhancing or increasing theTh-1 type response or by decreasing or diminishing the Th-2 typeresponse. Enhancing or increasing a Th1-type immune response may includeincreasing the production of cytokines such as IFNγ and/or TNFα and/orstimulating a cell-mediated immune response, such as the proliferationand activation of T-cells and/or macrophages specific for the antigen.Decreasing the Th2 immune response may include reducing theantibody-mediated, humoral immune responses and/or the production ofinterleukins 4, 5, and 13. In this respect, the invention also providesa method of shifting the Th-1/Th-2 balance of an immune response of ahost towards a Th-1 type immune response, which method comprisesadministering to the host at least one HMGN polypeptide in an amounteffective to shift the Th-1/Th-2 balance of an immune response of a hosttowards a Th-1 type immune response.

The antigen-specific immune response also can be enhanced by increasingor enhancing the activation or recruitment of dendritic cells. Thus, theinvention provides, as a related aspect, a method of enhancing theactivation or recruitment of dendritic cells in a host comprisingadministering to the host at least one HMGN polypeptide in an amounteffective to enhance the activation or recruitment of dendritic cells inthe host.

Activation of dendritic cells includes stimulating the maturation and/orthe migration of dendritic cells to a specific locale (e.g., the site ofan antigen or the site of chemotactic cytokine production, such as CCL2,CCL5, CCL19, CCL20, CCL21, etc.) and/or stimulating the maturation ofdendritic cells. The activation of dendritic cells can be detected ormeasured by the production of cytokines associated with the activationof dendritic cells. In particular, the HMGN polypeptide (or a functionalfragment thereof) may stimulate the dendritic cells to produce cytokinessuch as, for example, any or all of interleukin (IL)-6, IL-2, IL-8,IL-12, (e.g., IL-12p70), IL-1 (e.g., IL-1β), IL-10, IL-18, IL-23, tumornecrosis factors (TNF) (e.g., TNFα), and/or chemokines (e.g.,keratinocyte chemoattractant (KC), CXCL8, CCL1, CCL2, CCL5, CCL7, CCL8,CCL13, CCL17, CCL18, CCL20, and/or CCL22). Activation of dendritic cellscan also be detected or measured by the phosphorylation ofmitogen-activated protein kinases (MAPKs) associated with the activationof dendritic cells. In particular, the HMGN polypeptide (or a functionalfragment thereof) may stimulate the phosphorylation of MAPKs such as,for example, any or all of p44/42 MAPKs, p38 MAPKs, and/or c-JunN-terminal kinase (JNK) MAPKs. In addition, the activation of dendriticcells can be detected or measured by the activation of nuclear factorkappa-light-chain-enhancer of activated B cells (NF-κB). In particular,the HMGN polypeptide (or a functional fragment thereof) may stimulatethe activation of NF-κB and/or a decrease of I-κBα. Maturation ofdendritic cells can be detected or measured on the basis of theexpression of surface molecules that appear on mature dendritic cells.For example, mature dendritic cells typically express receptors thatenable them to respond to chemokines produced by the lymph node (e.g.,CCR7) and costimulatory (e.g., CD80, CD83, and CD86) and MHC (e.g.,HLA-ABC and HLA-DR) molecules that assist in activating T-cells.Maturation of dendritic cells also can be detected by a cell shapehaving veils and elongated dendrites, increased motility towardchemokines (e.g., CCL19 and CCL21), or reduced capacity for endocytosis.Maturation of dendritic cells also can be detected indirectly bymeasuring the capacity of dendritic cells to stimulate the proliferationor differentiation of naïve T-cells. Recruitment of dendritic cells canbe measured or detected by the movement of dendritic cells to a givenlocale. Assays for measuring or detecting an increase in the activationand/or recruitment of dendritic cells are known in the art and describedherein.

The HMGN polypeptides (including functional fragments thereof) can beobtained by methods known in the art. Suitable methods of de novosynthesizing polypeptides are described in references, such as Chan etal., Fmoc Solid Phase Peptide Synthesis, Oxford University Press,Oxford, United Kingdom, 2005; Peptide and Protein Drug Analysis, ed.Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood etal., Oxford University Press, Oxford, United Kingdom, 2000; and U.S.Pat. No. 5,449,752. Also, HMGN polypeptides (including functionalfragments) and antigens can be recombinantly produced using the nucleicacids described herein and standard recombinant methods. See, forinstance, Sambrook et al., Molecular Cloning: A Laboratory Manual,3^(rd) ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; andAusubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and John Wiley & Sons, NY, 1994. Further, the HMGNpolypeptides (including functional fragments thereof) can be isolatedand/or purified from a natural source, e.g., a human. Methods ofisolation and purification are well-known in the art. In this respect,the HMGN polypeptides are exogenous and can be synthetic, recombinant,or of natural origin.

The functional fragment of the HMGN polypeptide can comprise anycontiguous part of the HMGN polypeptide that retains a relevantbiological activity of the HMGN polypeptide, e.g., enhances anantigen-specific immune response. Any given fragment of an HMGNpolypeptide can be tested for such biological activity using methodsdescribed herein or otherwise known in the art. For example, thefunctional fragment can comprise, consist essentially of, or consist ofthe N-terminal nucleosomal binding domain (NBD) of the HMGN polypeptide(e.g., the sequence from ¹⁴Lys to ⁴⁹Lys of HMGN1 (SEQ ID NO: 11), thesequence from ²⁰Lys to ⁴⁹Lys of HMGN3a/3b (SEQ ID NO: 12), the sequencefrom ¹⁸Lys to ⁴⁷Lys of HMGN4 (SEQ ID NO: 13), and/or the sequence from¹⁷Lys to ⁴⁶Lys of Nsbp1 (SEQ ID NO: 14)). In reference to the parentHMGN polypeptide, the functional fragment preferably comprises, forinstance, about 10% or more, 25% or more, 30% or more, 50% or more, 60%or more, 80% or more, 90% or more, or even 95% or more of the parentHMGN polypeptide.

The HMGN polypeptides (including functional fragments) can beglycosylated, amidated, carboxylated, phosphorylated, esterified,N-acylated, cyclized via, e.g., a disulfide bridge, or converted into anacid addition salt and/or optionally dimerized or polymerized, orconjugated. Suitable pharmaceutically acceptable acid addition saltsinclude those derived from mineral acids, such as hydrochloric,hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids,and organic acids, such as tartaric, acetic, citric, malic, lactic,fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids,for example, p-toluenesulphonic acid.

Of course, the method of the invention can comprise administering two ormore HMGN polypeptides or functional fragment thereof, any of which maybe the same or different from one another. Furthermore, the HMGNpolypeptide or functional fragment thereof can be provided as part of alarger polypeptide construct. For instance, the HMGN polypeptide orfunctional fragment thereof can be provided as a fusion proteincomprising an HMGN polypeptide or functional fragment along with otheramino acid sequences or a nucleic acid encoding same. By way of furtherillustration, the HMGN polypeptide or functional fragment can beprovided by two or more fragments of an HMGN polypeptide (e.g., two ormore NBD domains, or at least one of each of the NBD domains), with orwithout a linking amino acid sequence and/or flanking sequences. TheHMGN polypeptide or fragment thereof also can be provided as part of aconjugate or nucleic acid encoding same. Conjugates, as well as methodsof synthesizing conjugates in general, are known in the art (See, forinstance, Hudecz, F., Methods Mol. Biol. 298: 209-223 (2005) and Kirinet al., Inorg Chem. 44(15): 5405-5415 (2005)).

The antigen can be any antigen against which an antigen-specific immuneresponse is desired. In some embodiments, the antigen is a microbialantigen. The microbial antigen can be a bacterial (e.g., anthrax,tuberculosis, etc.) antigen or a viral (e.g., influenza, humanimmunodeficiency virus (HIV), etc.) antigen. Microbial antigens aremolecules (e.g., polypeptide, lipid, carbohydrate, etc.) that areuniquely expressed by microbes, or greatly over-expressed by microbes ascompared to non-microbes, such that an immune response to the antigenresults in the more rapid destruction of the microbe as compared tonon-microbes.

Preferably, the antigen-specific immune response is an immune responseto a tumor antigen. Tumor antigens are molecules (e.g., polypeptide,lipid, carbohydrate, etc.) that are uniquely expressed by tumor cells,or greatly over-expressed by tumor cells as compared to non-tumor cells,such that an immune response to the antigen results in the more rapiddestruction of tumor cells as compared to normal (non-cancerous) cells.

The tumor antigen can be an antigen expressed by any cell of any canceror tumor. For example, the tumor antigen can be an antigen expressed byany cell of acute lymphocytic cancer, acute myeloid leukemia, alveolarrhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer ofthe anus, anal canal, or anorectum, cancer of the eye, cancer of theintrahepatic bile duct, cancer of the joints, cancer of the neck,gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear,cancer of the oral cavity, cancer of the vulva, chronic lymphocyticleukemia, chronic myeloid cancer, colon cancer, uterine cancer,esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor,lymphoid and other hematopoietic tumors, Hodgkin lymphoma, B celllymphoma, bronchial squamous cell cancer, hypopharynx cancer, kidneycancer, larynx cancer, liver cancer, pancreatic cancer, carcinoma, lungcancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynxcancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer,peritoneum, omentum, and mesentery cancer, pharynx cancer, prostatecancer, rectal cancer, renal cancer (e.g., renal cell carcinoma (RCC)),small intestine cancer, soft tissue cancer, stomach cancer, testicularcancer, thyroid cancer, ureter cancer, and urinary bladder cancer.

More specific examples of tumor antigens include polypeptides such asIg-idiotype of B cell lymphoma, mutant cyclin-dependent kinase 4 ofmelanoma, Pmel-17 (gp100) of melanoma, MART-1 (Melan-A) of melanoma, p15polypeptide of melanoma, tyrosinase of melanoma, MAGE 1, 2 and 3 ofmelanoma, thyroid medullary, small cell lung cancer, colon and/orbronchial squamous cell cancer, BAGE of bladder, melanoma, breast, andsquamous-cell carcinoma, gp75 of melanoma, oncofetal antigen ofmelanoma; carbohydrate/lipids such as muci mucin of breast, pancreas,and ovarian cancer, GM2 and GD2 gangliosides of melanoma; oncogenes suchas mutant p53 of carcinoma, mutant ras of colon cancer and HER21neuproto-onco-gene of breast carcinoma; viral products such as humanpapilloma virus polypeptides of squamous cell cancers of cervix andesophagus.

A method of the invention may further comprise administering an antigento the host, especially a tumor antigen. The antigen can be any of thosediscussed above with respect to the antigen-specific immune response. Ofcourse, two or more different antigens can be administered to the host.

When an antigen is administered in connection with a method of theinvention, the antigen and HMGN polypeptide (or functional fragmentthereof) can be administered simultaneously (as a single composition orin different compositions) or sequentially in any order. The methods mayinclude administering the antigen first, followed by the HMGNpolypeptide (or functional fragment thereof), or the method may includeadministering the HMGN polypeptide (or functional fragment thereof)first, followed by the antigen. Regardless of the order of theadministration of the HMGN polypeptide (or functional fragment thereof)and the antigen, the HMGN polypeptide (or functional fragment thereof)and the antigen are preferably administered in close enough successionto enhance an immune response against the antigen.

The HMGN polypeptide (or functional fragment thereof) and the antigenalso can be part of a fusion protein. The fusion protein can compriseone or more HMGN polypeptides (or functional fragments thereof) and/orone or more antigens. Suitable methods of making fusion proteins areknown in the art, and include, for example, recombinant methods. See,for instance, Choi et al., Mol. Biotechnol. 31: 193-202 (2005). In otherembodiments, the HMGN polypeptide, including any of the functionalfragments thereof, may be provided as a conjugate with the antigen.Conjugates, as well as methods of synthesizing conjugates in general,are known in the art (See, for instance, Hudecz, F., Methods Mol. Biol.298: 209-223 (2005) and Kirin et al., Inorg Chem. 44(15): 5405-5415(2005)).

The HMGN polypeptide or fragment thereof and the antigen, when used, canbe administered to the host by administering a nucleic acid encodingsuch molecules to the host. “Nucleic acid” as used herein includes“polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” andgenerally means a polymer of DNA or RNA, which can be single-stranded ordouble-stranded, synthesized or obtained (e.g., isolated and/orpurified) from natural sources, which can contain natural, non-naturalor altered nucleotides, and which can contain a natural, non-natural oraltered internucleotide linkage, such as a phosphoroamidate linkage or aphosphorothioate linkage, instead of the phosphodiester found betweenthe nucleotides of an unmodified oligonucleotide.

Nucleic acids encoding the HMGN polypeptides and antigens discussedherein are known in the art (e.g., SEQ ID NOs: 6-10 and degeneratenucleic acid sequences encoding the same amino acid sequences), and canbe constructed based on chemical synthesis and/or enzymatic ligationreactions using procedures known in the art. See, for example, Sambrooket al., supra, and Ausubel et al., supra. For example, a nucleic acidcan be chemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed upon hybridization (e.g., phosphorothioate derivatives andacridine substituted nucleotides).

The nucleic acids can be incorporated into a recombinant expressionvector. For purposes herein, the term “recombinant expression vector”means a genetically-modified oligonucleotide or polynucleotide constructthat permits the expression of an mRNA or polypeptide by a host cell,when the construct comprises a nucleotide sequence encoding the mRNA orpolypeptide, and the vector is contacted with the cell under conditionssufficient to have the mRNA or polypeptide expressed within the cell.The vectors are not naturally-occurring as a whole. However, parts ofthe vectors can be naturally-occurring. The recombinant expressionvectors can comprise any type of nucleotides, including, but not limitedto DNA and RNA, which can be single-stranded or double-stranded,synthesized or obtained in part from natural sources, and which cancontain natural, non-natural or altered nucleotides. The recombinantexpression vectors can comprise naturally-occurring ornon-naturally-occurring internucleotide linkages, or both types oflinkages. Preferably, the non-naturally occurring or altered nucleotidesor internucleotide linkages does not hinder the transcription orreplication of the vector.

The recombinant expression vector can be any suitable recombinantexpression vector, and can be used to transform or transfect anysuitable host. Suitable vectors include those designed for propagationand expansion or for expression or both, such as plasmids and viruses.The vector can be of the pUC series (Fermentas Life Sciences), thepBluescript series (Stratagene, LaJolla, Calif.), the pET series(Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala,Sweden), or the pEX series (Clontech, Palo Alto, Calif.). Bacteriophagevectors, such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149,also can be used. Examples of plant expression vectors include pBI01,pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animalexpression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech).Preferably, the recombinant expression vector is a viral vector, e.g., aretroviral vector.

The recombinant expression vectors can be prepared using standardrecombinant DNA techniques described in, for example, Sambrook et al.,supra, and Ausubel et al., supra. Constructs of expression vectors,which are circular or linear, can be prepared to contain a replicationsystem functional in a prokaryotic or eukaryotic host cell. Replicationsystems can be derived, e.g., from ColE1, 2μ plasmid, λ, SV40, bovinepapilloma virus, and the like.

Desirably, the recombinant expression vector comprises regulatorysequences, such as transcription and translation initiation andtermination codons, which are specific to the type of host (e.g.,bacterium, fungus, plant, or animal) into which the vector is to beintroduced, as appropriate and taking into consideration whether thevector is DNA- or RNA-based.

The recombinant expression vector can include one or more marker genes,which allow for selection of transformed or transfected hosts. Markergenes include biocide resistance, e.g., resistance to antibiotics, heavymetals, etc., complementation in an auxotrophic host to provideprototrophy, and the like. Suitable marker genes for the inventiveexpression vectors include, for instance, neomycin/G418 resistancegenes, hygromycin resistance genes, histidinol resistance genes,tetracycline resistance genes, and ampicillin resistance genes.

The recombinant expression vector can comprise a native or normativepromoter and/or stop codon operably linked to the nucleotide sequenceencoding the HMGN polypeptide (including functional fragments thereof),or to the nucleotide sequence which is complementary to the nucleotidesequence encoding the HMGN polypeptide or functional fragment thereof.The selection of stop codons and promoters, e.g., strong, weak,inducible, tissue-specific and developmental-specific, is within theordinary skill of the artisan. Similarly, the combining of a nucleotidesequence with a stop codon and a promoter is also within the skill ofthe artisan. The promoter can be a non-viral promoter or a viralpromoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, anRSV promoter, and a promoter found in the long-terminal repeat of themurine stem cell virus.

The HMGN polypeptides (including functional fragments thereof) andnucleic acids encoding such peptides can be of synthetic or naturalorigin, and can be isolated or purified to any degree. The terms“isolated” and “purified” as used herein means having been increased inpurity, wherein “purity” is a relative term, and not to be necessarilyconstrued as absolute purity. For example, the purity can be at leastabout 50%, can be greater than 60%, 70% or 80%, or can be 100%.

The methods described herein may be used for any purpose, e.g., thetreatment or prevention of disease, especially cancer. Exemplary cancersthat may be treated or prevented using the methods described herein mayinclude any of those discussed above with respect to the tumor antigens.

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount of any level of treatment or preventionof cancer in a mammal. Furthermore, the treatment or prevention providedby the inventive method can include treatment or prevention of one ormore conditions or symptoms of the disease, e.g., cancer, being treatedor prevented. Also, for purposes herein, “prevention” can encompassdelaying the onset of the disease, or a symptom or condition thereof.With respect to the inventive methods, the cancer can be any cancer,including any of the cancers associated with any of the tumor antigensdescribed herein.

For purposes of the invention, the amount or dose of the HMGN materialadministered should be sufficient to effect the desired biologicalresponse, e.g., a therapeutic or prophylactic response, in the subjector animal over a reasonable time frame. The dose will be determined bythe efficacy of the particular HMGN material and the condition of thehost (e.g., human), as well as the body weight of the host (e.g., human)to be treated. The dose of the HMGN material also will be determined bythe existence, nature and extent of any adverse side effects that mightaccompany the administration of a particular HMGN material. Typically,the attending physician will decide the dosage of the HMGN material withwhich to treat each individual patient, taking into consideration avariety of factors, such as age, body weight, general health, diet, sex,HMGN material to be administered, route of administration, and theseverity of the condition being treated.

The host referred to in the inventive methods can be any host capable ofexhibiting an antigen-specific immune response. Preferably, the host isa mammal. As used herein, the term “mammal” refers to any mammal,including, but not limited to, mammals of the order Rodentia, such asmice and hamsters, and mammals of the order Logomorpha, such as rabbits.It is preferred that the mammals are from the order Carnivora, includingFelines (cats) and Canines (dogs). It is more preferred that the mammalsare from the order Artiodactyla, including Bovines (cows) and Swines(pigs) or of the order Perssodactyla, including Equines (horses). It ismost preferred that the mammals are of the order Primates, Ceboids, orSimoids (monkeys) or of the order Anthropoids (humans and apes). Anespecially preferred mammal is the human. The host can be non-diseased,a host afflicted with a disease, such as cancer, or a host predisposedto a disease, such as cancer.

The invention also provides a pharmaceutical composition comprising (a)an HMGN polypeptide comprising HMGN1 (SEQ ID NO: 1), HMGN3a (SEQ ID NO:2), HMGN3b (SEQ ID NO: 3), HMGN4 (SEQ ID NO: 4), Nsbp1 (NBD-45) (SEQ IDNO: 5), or functional fragment thereof, and (b) an antigen, especially atumor antigen. Alternatively, the pharmaceutical composition comprises(a) a nucleic acid encoding an HMGN polypeptide comprising HMGN1 (SEQ IDNO: 1), HMGN3a (SEQ ID NO: 2), HMGN3b (SEQ ID NO: 3), HMGN4 (SEQ ID NO:4), Nsbp1 (NBD-45) (SEQ ID NO: 5), or functional fragment thereof, and(b) a nucleic acid encoding an antigen, especially a tumor antigen. Thepharmaceutical composition can, of course, comprise more than one HMGNpolypeptide or fragment thereof (e.g., two or more different HMGNpolypeptides) or one or more nucleic acids encoding more than one HMGNpolypeptide or fragment thereof (e.g., two or more different HMGNpolypeptides). Alternatively or in addition, the pharmaceuticalcomposition can comprise more than one tumor antigen (e.g., two or moredifferent antigens) or one or more nucleic acids encoding more than onetumor antigen (e.g., two or more different antigens). All other featuresof the HMGN polypeptides (including functional fragments thereof),antigens, tumor antigens, and nucleic acids are as described withrespect to the methods of the invention.

The pharmaceutical composition can comprise other active ingredients inaddition to the HMGN polypeptide, fragment thereof, and tumor antigen.For example, the pharmaceutical composition can comprise otherpharmaceutically active agents or drugs, such as a chemotherapeuticagent, e.g., asparaginase, busulfan, carboplatin, cisplatin,daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea,methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.;biological response modifiers, e.g., bacillus calmette-guerin (BCG),etc.; cytokines, e.g., IL-2, IFNs, GM-CSF, etc.; and/or antibodies,e.g., trastuzumab (Herceptin®, available from Genentech, South SanFrancisco, Calif., U.S.A.), etc.

The pharmaceutical composition typically will comprise a carrier.Preferably, the carrier is a pharmaceutically acceptable carrier. Withrespect to pharmaceutical compositions, the carrier can be any of thoseconventionally used and is limited only by chemico-physicalconsiderations, such as solubility and lack of reactivity with theactive compound(s), and by the route of administration. Thepharmaceutically acceptable carriers described herein, for example,vehicles, excipients, and diluents, are well-known to those skilled inthe art and are readily available to the public. It is preferred thatthe pharmaceutically acceptable carrier be one which is chemically inertto the active agent(s) and one which has no detrimental side effects ortoxicity under the conditions of use. The choice of carrier will bedetermined in part by the particular compounds used in thepharmaceutical composition, as well as by the particular method used toadminister the HMGN material.

The following formulations for oral, intravenous, intramuscular,subcutaneous, or intraperitoneal administration are exemplary and are inno way limiting. More than one route can be used to administer the HMGNmaterials and/or tumor antigen, and in certain instances, a particularroute can provide a more immediate and more effective response thananother route.

Oral formulations may include any suitable carrier. For example,formulations suitable for oral administration may comprise suitablecarriers, such as lactose, sucrose, starch, talc magnesium stearate,crystalline cellulose, methyl cellulose, carboxymethyl cellulose,glycerin, sodium alginate or gum arabic among others.

Intravenous, intramuscular, subcutaneous, or intraperitonealformulations may include any suitable carrier. For example, formulationssuitable for intravenous, intramuscular, subcutaneous, orintraperitoneal administration may comprise sterile aqueous solutions ofthe HMGN polypeptide (or functional fragment thereof) and/or the tumorantigen with solutions which are preferably isotonic with the blood ofthe recipient. Such formulations may be prepared by dissolving the HMGNpolypeptide (or functional fragment thereof) and/or the tumor antigen inwater containing physiologically compatible substances such as sodiumchloride (e.g. 0.1-2.0M), glycine, and the like, and having a bufferedpH compatible with physiological conditions to produce an aqueoussolution, and rendering said solution sterile.

For purposes of the invention, the amount or concentration of the HMGNpolypeptide or fragment thereof, tumor antigen, and other optionalactive ingredients used in the pharmaceutical composition should besufficient to effect a desired biological response, e.g., a therapeuticor prophylactic response, in the subject or animal using a reasonabledosage regimen over a reasonable time frame. For example, theconcentration of the HMGN polypeptide or fragment thereof, tumorantigen, and other optional active ingredients should be sufficient toenhance an antigen-specific immune response as defined herein withrespect to the methods of the invention.

The pharmaceutical compositions of the invention may be used for anypurpose, but are thought to be especially useful in conjunction with themethods of the invention and for the treatment or prevention of disease,such as cancer. Exemplary cancers that may be treated or preventedinclude acute lymphocytic cancer, acute myeloid leukemia, alveolarrhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer ofthe anus, anal canal, or anorectum, cancer of the eye, cancer of theintrahepatic bile duct, cancer of the joints, cancer of the neck,gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear,cancer of the oral cavity, cancer of the vulva, chronic lymphocyticleukemia, chronic myeloid cancer, colon cancer, uterine cancer,esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor,lymphoid and other hematopoietic tumors, Hodgkin lymphoma, B celllymphoma, bronchial squamous cell cancer, hypopharynx cancer, kidneycancer, larynx cancer, liver cancer, pancreatic cancer, carcinoma, lungcancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynxcancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer,peritoneum, omentum, and mesentery cancer, pharynx cancer, prostatecancer, rectal cancer, renal cancer (e.g., renal cell carcinoma (RCC)),small intestine cancer, soft tissue cancer, stomach cancer, testicularcancer, thyroid cancer, ureter cancer, and urinary bladder cancer.

The pharmaceutical compositions of the invention may advantageously benontoxic. Without being bound to any particular theory, it is believedthat because the HMGN polypeptides are endogenous molecules, theadministration of an HMGN polypeptide may not cause toxic effects inmammals, particularly, humans.

The invention also provides a method of suppressing an antigen-specificimmune response in a host. The method comprises administering to thehost an HMGN polypeptide antagonist, wherein the HMGN polypeptidecomprises HMGN1 (SEQ ID NO: 1), HMGN3a (SEQ ID NO: 2), HMGN3b (SEQ IDNO: 3), HMGN4 (SEQ ID NO: 4), or Nsbp1 (SEQ ID NO: 5), in an amounteffective to suppress the immune response.

An immune response is suppressed in accordance with the invention if theimmune response is diminished, quantitatively or qualitatively, afteradministration of an HMGN polypeptide antagonist as compared to theimmune response in the absence of the administration of an HMGNpolypeptide antagonist. A quantitative decrease in an immune responseencompasses a decrease in the magnitude or degree of the response. Themagnitude or degree of an immune response can be measured on the basisof any number of known parameters, such as a decrease in the level ofcytokine (e.g., antigen-specific cytokine) production (cytokineconcentration), a decrease in the number of lymphocytes activated (e.g.,proliferation of lymphocytes (e.g., antigen-specific lymphocytes)) orrecruited, and/or a decrease in the production of antibodies(antigen-specific antibodies) (antibody concentration), etc. Aqualitative decrease in an immune response encompasses any change in thenature of the immune response that renders it less effective atmediating the destruction of a given antigen. For the purposes of thisinvention, the quality of an immune response is considered diminished ifthe relative balance of the immune response is shifted towards the Th-2type immune response and away from the Th-1 type immune response. Therelative balance of an immune response may be distinguished and measuredby methods known in the art and as described herein. For example, ashift toward the Th-2 type response may be characterized by an increasein IL-4, IL-5, and/or IL-13 and no increase or a reduced increase inIFNγ. Conversely, a shift toward the Th-1 type response may becharacterized by an increase in IFNγ and no increase or a reducedincrease in IL-4, IL-5, and/or IL-13. Another exemplary method mayinclude measuring the subtypes of antigen-specific IgG antibodiesproduced during an immune response. A lower level (concentration) ofIgG2a antibodies versus IgG1 antibodies suggests a Th2-type immuneresponse. Conversely, a lower level (concentration) of IgG1 antibodiesversus IgG2a antibodies suggests a Th1-type immune response. Qualitativeand quantitative diminishment of an immune response can occursimultaneously, and are not mutually exclusive.

Preferably, the immune response is suppressed by shifting the Th-1/Th-2balance of an immune response towards a Th-2 type response and away froma Th-1 type response, i.e., by suppressing or decreasing the Th-1 typeresponse or by increasing or enhancing the Th-2 type response.Suppressing or decreasing a Th1-type immune response may includedecreasing the production of cytokines such as IFNγ and/or TNFα and/orreducing a cell-mediated immune response, such as the proliferation andactivation of T-cells and/or macrophages specific for the antigen.Decreasing the Th1 immune response may include increasing theantibody-mediated, humoral immune responses and/or the production ofinterleukins 4, 5, and 13.

The immune response also can be suppressed by decreasing or suppressingthe activation or recruitment of dendritic cells. Suppressing theactivation of dendritic cells includes reducing the maturation and/orthe migration of dendritic cells, e.g., to a specific locale (e.g., thesite of an antigen or the site of chemotactic cytokine production, suchas CCL2, CCL5, CCL19, CCL20, CCL21, etc.). Suppressing the activation ofdendritic cells can be measured by the lack of production of cytokinesassociated with the activation of dendritic cells. In particular, theHMGN polypeptide antagonist may suppress the dendritic cell productionof cytokines such as, for example, any or all of interleukin (IL)-6,IL-8, IL-12, (e.g., IL-12p70), IL-1 (e.g., IL-1β), IL-10, IL-18, IL-23,tumor necrosis factors (TNF) (e.g., TNFα), and/or chemokines (e.g.,CXCL8, CCL1, CCL2, CCL5, CCL7, CCL8, CCL13, CCL17, CCL18, CCL20, and/orCCL22). The lack of mature dendritic cells, or a reduction in maturedendritic cells, can be detected or measured on the basis of the lack ofexpression of surface molecules that appear on mature dendritic cells.For example, immature dendritic cells typically do not express receptorsthat enable them to respond to chemokines produced by the lymph node(e.g., CCR7) or costimulatory (e.g., CD80, CD83, and CD86) or MHC (e.g.,HLA-ABC and HLA-DR) molecules that assist in activating T-cells.Immature dendritic cells also can be detected by a cell shape lackingveils and elongated dendrites, decreased motility toward chemokines(e.g., CCL19 and CCL21), or increased capacity for endocytosis. Immaturedendritic cells can also be detected indirectly by measuring theinability of dendritic cells to stimulate the proliferation ordifferentiation of naïve T-cells. The absence of recruitment ofdendritic cells can be measured or detected by a lack of movement ofdendritic cells to a given locale. Assays for measuring or detecting adecrease in the activation and/or recruitment of dendritic cells areknown in the art and described herein.

The HMGN antagonist can be any agent that inhibits the biologicalactivity of an HMGN polypeptide. Inhibition of an HMGN polypeptide maybe characterized by suppression of an immune response in any of the waysdescribed herein. The HMGN antagonists include agents that bind to theHMGN polypeptide or functional fragment thereof (e.g., the NBD of theHMGN polypeptide), thereby inhibiting its function, as well as agentsthat compete with the HMGN polypeptide or functional fragment thereof(e.g., the NBD of the HMGN polypeptide) for the native HMGN bindingsite. By way of illustration, the HMGN antagonist can be an antibody orantibody fragment, an antisense nucleotide, or a chemical inhibitor(e.g., small molecule or peptide inhibitor).

Anti-HMGN antibodies and antibody fragments can be monoclonal orpolyclonal. Anti-HMGN antibodies and antibody fragments can be preparedusing the HMGN proteins disclosed herein and routine techniques.Examples of such antibodies or antibody fragments include those specificto a functional domain of HMGN (e.g., nucleosomal binding domain).

Antisense nucleic acid (e.g., RNA or DNA) include, for example,interfering nucleic acids such as RNAi and siRNA molecules. Suchantisense nucleic acids are commercially available and can be preparedusing the nucleic acid sequences encoding the HMGN polypeptidesdisclosed herein and routine techniques.

Chemical inhibitors of HMGN include small molecules and peptides thatbind the HMGN polypeptide or functional fragment thereof or compete withthe HMGN polypeptide or functional fragment thereof for its nativebinding site. Suitable inhibitors can include, for example, a non-activefragment or mutant of an HMGN polypeptide. Chemical inhibitors of HMGNcan be identified using routine techniques. For example, chemicalinhibitors can be tested in binding assays to identify molecules andpeptides that bind to a given HMGN polypeptide or functional fragmentthereof with sufficient affinity to inhibit HMGN biological function.Also, competition assays can be performed to identify small-moleculesand peptides that compete with HMGN or functional fragment thereof forbinding to its native binding site. Such techniques could be used inconjunction with mutagenesis of the HMGN polypeptide or functionalfragment thereof itself, and/or with high-throughput screens of knownchemical inhibitors.

The methods of suppressing an immune response described herein may beused for any purpose, e.g., the treatment or prevention of a diseaseassociated with a heightened or Th-1 polarized immune response,particularly any disease that may be effectively treated or prevented byshifting the Th-1/Th-2 balance of an immune response away from aTh1-type response and toward a Th2-type response. Exemplary diseasesthat may be treated or prevented using the methods of suppressing animmune response described herein include parasitic infections (e.g.,Giardia intestinalis, Trichomonas vaginalis, Cryptosporidium, Toxoplasmagondii and Leishmania major) and inflammatory or autoimmune disorders(e.g., atherosclerosis; asthma; lung fibrosis; bronchitis; respiratorydistress syndrome; obstructive pulmonary disease; allergies; multiplesclerosis; dermatitis; psoriasis; gastroenteritis; colitis (e.g.,ulcerative colitis); Crohn's disease; cystic fibrosis; celiac disease;inflammatory bowel disease; conjunctivitis; uveitis; autoimmune kidneydisease; diabetic nephropathy; cachexia; coronary restenosis; sinusitis,cystitis; urethritis; serositis; uremic pericarditis; cholecystis;vaginitis; drug reactions; hepatitis; pelvic inflammatory disease;multiple myeloma; vitiligo; alopecia; Addison's disease; Hashimoto'sdisease; Graves disease; atrophic gastritis/pernicious anemia; acquiredhypogonadism/infertility; hypoparathyroidism; multiple sclerosis;Myasthenia gravis; Coombs positive hemolytic anemia; systemic lupuserthymatosis; Siogren's syndrome, rheumatoid arthritis; endotoxemia; andimmune mediated (type-1) diabetes).

For purposes of the invention, the amount or dose of the HMGNpolypeptide antagonist administered should be sufficient to effect thedesired biological response, e.g., a therapeutic or prophylacticresponse, in the subject or animal over a reasonable time frame. Thedose will be determined by the efficacy of the particular HMGNpolypeptide antagonist and the condition of the host (e.g., human), aswell as the body weight of the host (e.g., human) to be treated. Thedose of the HMGN polypeptide antagonist also will be determined by theexistence, nature and extent of any adverse side effects that mightaccompany the administration of a particular HMGN polypeptideantagonist. Typically, the attending physician will decide the dosage ofthe HMGN polypeptide antagonist with which to treat each individualpatient, taking into consideration a variety of factors, such as age,body weight, general health, diet, sex, HMGN polypeptide antagonist tobe administered, route of administration, and the severity of thecondition being treated.

Carriers, formulations, and routes of administration of the HMGNpolypeptide antagonist may be any of those described herein for theadministration of the HMGN polypeptide.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLE 1

This example demonstrates that HMGN1 (SEQ ID NO: 1) induces dendriticcells to produce cytokines in a dose-dependent manner.

Human dendritic cells were cultured for 24 hours in RPMI 1640 medium,which contains 10% fetal bovine serum, 50 ng/mL recombinant human (rh)GM-CSF, 50 ng/mL rhIL-4. The cultures were treated with either HMGN1(SEQ ID NO: 1) or HMGN2 at concentrations of 0, 0.2, 1, or 5 μg/mL.Untreated culture (sham) served as a control. The supernatants of thedendritic cell cultures were assayed for IL-6, IL-8, IL-12p70, and TNFαconcentration by cytokine array. Experiments were repeated three timesand the average (mean±SD) determined. The results are presented in FIG.1A.

As shown in FIG. 1A, HMGN1 (SEQ ID NO: 1) stimulated the production ofIL-6, IL-8, IL-12p70, and TNFα in dendritic cells a dose-dependentmanner, whereas HMGN2 and untreated cultures showed no significantproduction of these cytokines. These results support the use of HMGN1(SEQ ID NO: 1) to activate and/or recruit dendritic cells and enhance animmune response.

EXAMPLE 2

This example demonstrates that HMGN1 (SEQ ID NO: 1) induces dendriticcells to produce cytokines in a time-dependent manner.

Human dendritic cells were cultured for 24 hours in RPMI 1640 medium.The cultures were treated with 1 μg/mL of HMGN1 (SEQ ID NO: 1) or HMGN2,and the supernatents were analyzed by cytokine array for IL-6, IL-8,IL-12p70, and TNFα concentration at 6, 24, and 48 hours. Untreatedculture (sham) served as a control. Experiments were repeated threetimes and the average (mean±SD) determined. The results are presented inFIG. 1B.

As shown in FIG. 1B, HMGN1 (SEQ ID NO: 1) stimulates the production ofIL-6, IL-8, IL-12p70, and TNFα in a time-dependent manner, whereas HMGN2and untreated cultures showed no significant production of thesecytokines. These results support the use of HMGN1 (SEQ ID NO: 1) toactivate and/or recruit dendritic cells and enhance an immune response.

EXAMPLE 3

This example demonstrates that HMGN1 (SEQ ID NO: 1) upregulatesdendritic cell expression of surface costimulatory molecules and surfaceMHC molecules.

Human dendritic cells were cultured for 48 hours at 37° C. in a CO₂incubator in RPMI 1640 medium. Cultures were treated with 1 μg/mL ofHMGN1 (SEQ ID NO: 1) or HMGN2. Culture treated with 1 μg/mLlipopolysaccharide (LPS) and untreated culture (sham) served as positiveand negative controls, respectively. The dendritic cells wereimmunostained and analyzed for the expression of surface molecules byflow cytometry. The results are presented in FIG. 2, wherein open-areacurves represent staining with isotype-matched control antibody, andshaded-area curves represent staining with antibodies against thevarious surface molecules.

As shown in FIG. 2, treatment with HMGN1 (SEQ ID NO: 1) inducessignificantly greater expression of costimulatory molecules (CD80, CD83,and CD86) and MHC molecules (HLA-ABC and HLA-DR) as compared totreatment with HMGN2 or without treatment. These results support the useof HMGN1 (SEQ ID NO: 1) to activate dendritic cells and enhance animmune response.

EXAMPLE 4

This example demonstrates that HMGN1 (SEQ ID NO: 1) enhances theantigen-presenting capacity of human dendritic cells.

Human dendritic cells were cultured with RPMI 1640 medium for 48 hours.Cultures were treated with either HMGN1 (SEQ ID NO: 1) (1 μg/mL) orHMGN2 (1 μg/mL). Culture treated with LPS (1 μg/mL) (as a positivecontrol) and untreated culture (sham) served as positive and negativecontrols, respectively. The cultured cells were then used to stimulatethe proliferation of allogeneic human T cells (10⁵) in a mixedlymphocyte reaction. The proliferation of allogeneic T cells wasmeasured as a function of tritiated thymidine (³H-TdR) incorporation.The results are presented in FIG. 3.

As shown in FIG. 3, dendritic cells treated with HGMN1 stimulated theproliferation of allogeneic T cells to a degree equal to or greater thanthat demonstrated by the positive control. HMGN2-treated culture and thenegative control showed no significant amount of T-cell proliferation.These results support the use of HMGN1 (SEQ ID NO: 1) to activatedendritic cells and enhance an immune response.

EXAMPLE 5

This example demonstrates that HMGN1 (SEQ ID NO: 1) stimulates thematuration of dendritic cells.

Human dendritic cells were cultured with RPMI 1640 medium for 48 hours.Cultures were treated with either HMGN1 (SEQ ID NO: 1) (1 μg/mL) orHMGN2 (1 μg/mL). Culture treated with LPS (1 μg/mL) (as a positivecontrol) and untreated culture (sham) served as positive and negativecontrols, respectively. The migratory response of the dendritic cellstoward CCL5, CCL21, and CXCL12 chemokines was measured according to thenumber of cells per high power field (No./HPF). The average number ofdendritic cells (mean±SD of triplicate wells) that migrated in responseto the chemokines is presented in FIG. 4.

As shown in FIG. 4, dendritic cells in the negative control culture(sham) migrated toward CCL5 and not CCL21, indicating immaturity.Conversely, dendritic cells cultured in the presence of HMGN1 (SEQ IDNO: 1) migrated toward CCL21 and not CCL5. These results indicate thatHMGN1 (SEQ ID NO: 1) treatment converted the dendritic cells fromCCL5-responsive to CCL21-responsive, which is characteristic ofdendritic cell maturation. The results support the use of HMGN1 (SEQ IDNO: 1) to activate dendritic cells and enhance an immune response.

EXAMPLE 6

This example demonstrates that HMGN1 (SEQ ID NO: 1) stimulates therecruitment of dendritic cells.

C57BL/6 mice (female, 3/group, 10 weeks old) were injectedintraperitoneally with PBS alone (control) or PBS containing 1 μg ofHMGN1 (SEQ ID NO: 1). After 4 hours, the cells in the peritoneal cavitywere washed out, stained with antibodies against surface markerscharacteristic of mouse dendritic cells (CD11c+, CD11c+/CD11b+,CD11c+/B220+, CD11c+/CD11b+/B220+), and analyzed by flow cytometry. Theresults are presented in FIG. 5.

As shown in FIG. 5, HMGN1 (SEQ ID NO: 1) treatment stimulated theaccumulation of various subpopulations of mouse dendritic cells (CD11c+,CD11c+/CD11b+, CD11c+/B220+, CD11c+/CD11b+/B220+) into the peritonealcavity. These results support the use of HMGN1 (SEQ ID NO: 1) to recruitdendritic cells and enhance an immune response in vivo.

EXAMPLE 7

This example demonstrates that HMGN1 (SEQ ID NO: 1) promotes anantigen-specific immune response.

C57BL/6 mice (female, 4/group, 8 weeks old) were intraperitoneallyimmunized with ovalbumin (OVA) alone, OVA mixed with alum (2.5 mg), orHMGN1 (SEQ ID NO: 1) (1 μg) on Day 1, booster immunized with OVA aloneon Day 14, and euthanized on Day 21. The splenocytes of immunized micewere stimulated in vitro with OVA (0, 2, 10, and 50 μg/ml) for 5 days tomeasure OVA-specific proliferation (FIG. 6A) or stimulated with 20 μg/mLof OVA for 3 days (FIG. 6B). Cytokine production was measured, and theresults are presented in FIGS. 6A and 6B.

As shown in FIG. 6A, the splenocytes of mice immunized with OVA plusHMGN1 (SEQ ID NO: 1) proliferated vigorously in a dose dependent mannerupon in vitro OVA stimulation, indicating that HMGN1 (SEQ ID NO: 1)promoted an OVA-specific immune response. As shown in FIG. 6B, thesplenocytes of mice immunized with OVA plus HMGN1 (SEQ ID NO: 1)produced significant amounts of IFNγ and TNFα, but not IL-4, indicatingthat HMGN1 (SEQ ID NO: 1) predominantly enhanced a Th1-type immuneresponse. Conversely, alum, which served as a control that predominantlyenhances a Th2 immune response, produced a high level of IL-4 and didnot increase IFNγ. These results support the use of HMGN1 (SEQ ID NO: 1)to enhance an antigen-specific immune response and to shift theTh-1/Th-2 balance of an immune response of a host towards a Th-1 typeimmune response.

EXAMPLE 8

This example demonstrates the importance of HMGN1 (SEQ ID NO: 1) to theantigen-specific immune response in vivo.

HMGN1 knockout (HMGN1−/−) and littermate-matched WT (HMGN1+/+) mice (4mice/group) were intra-peritoneally immunized on day 1 with ovalbumin(OVA) in the presence of alum or LPS, and boosted on day 14. On day 21,spleens of each group of immunized mice were pooled for the preparationof single splenocyte suspension. Subsequently, the splenocytes werestimulated in vitro with OVA for 48 hours and the cytokineconcentrations in the supernatants were measured by cytokine array.

As shown in FIGS. 7A and 7B, splenocytes from HMGN1 knockout miceproduced significantly less T cell cytokines (e.g., IL-4, IL-13, andIFNγ) as compared to wild-type mice, irrespective of adjuvant used (alumor LPS). These results show that HMGN1 (SEQ ID NO: 1) plays asignificant role in the generation of a T-cell immune response.

EXAMPLE 9

This example demonstrates that the administration of exogenous HMGN1(SEQ ID NO: 1) promotes an antigen-specific immune response in vivo.

C57BL/6 mice (4/group) were immunized with anthrax vaccine adsorbed(AVA) alone or in combination with 1-5 μg/mouse of HMGN1 (SEQ ID NO: 1)on day 1 and day 14 (boost). On day 10 and day 21, serum samples weretaken from all mice and anti-AVA specific antibody titers were measuredby ELISA.

As shown in FIGS. 8A and 8B, mice immunized with AVA+HMGN1 (SEQ IDNO: 1) (1 μg/mouse) produced a higher level of secondary (day 21)anti-AVA antibodies as compared to mice immunized with AVA only. HMGN1(SEQ ID NO: 1) at 5 μg/mouse (5-10 fold) enhanced both primary (day 10)and secondary (day 21) anti-AVA antibody responses. These resultssupport the use of HMGN1 (SEQ ID NO: 1) to enhance an antigen-specificimmune response.

EXAMPLE 10

This example demonstrates that HMGN1 (SEQ ID NO: 1) induces dendriticcells to produce cytokines.

Mice bone marrow-derived DCs were treated with various doses of HMGN1(SEQ ID NO: 1) for 24 hours and the production of various inflammatorycytokines in the supernatants were subsequently measured by cytokinearray.

As shown in FIGS. 9A and 9B, HMGN1 (SEQ ID NO: 1) stimulated theproduction of various inflammatory cytokines (TNFα, IL-1β, keratinocytechemoattractant (KC), IL-10, IL-6, and IL-12p70) by mouse DCs. Theresults support the use of HMGN1 (SEQ ID NO: 1) to activate and/orrecruit dendritic cells and modulate an immune response.

EXAMPLE 11

This example demonstrates to importance of HMGN1 (SEQ ID NO: 1) to theinflammatory immune response in vivo.

HMGN1 WT (HMGN1+/+) or KO (HMGN1−/−) mice (4 mice/group) were immunizedintraperitoneally with OVA alone or OVA in the presence of alum or LPS(endotoxin). At 24 or 96 hours after the immunization, mouse serumsamples were taken and various cytokines were measured.

As shown in FIG. 10A, HMGN1 KO mice failed to produce detectable levelsof the inflammatory cytokines tested (IL-1β, IL-2, IL-6, and IL-12p70)at 24 and 96 hours after immunization, and produced less TNFα at 96hours as compared to HMGN1 WT mice, irrespective of the adjuvant usedfor the immunization (either alum or LPS). These results demonstrate theimportance of HMGN1 (SEQ ID NO: 1) in the induction of inflammatorycytokines, and support the use of HMGN1 (SEQ ID NO: 1) as a basis tomodulate the inflammatory response, such as through the use of HMGN1(SEQ ID NO: 1) inhibitory molecules.

EXAMPLE 12

This example demonstrates that HMGN1 (SEQ ID NO: 1) stimulates theactivation of dendritic cells.

Mouse bone marrow-derived DCs were untreated or treated with HMGN1 (SEQID NO: 1) (at 1 μg/ml) at 37° C. for 20 or 60 minutes. At the end oftreatment, DCs were washed extensively with ice-cold PBS, pelleted, andsolubilized in sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) lysis buffer (at 10⁷/ml) to make cell lysate. DC lysates werethen loaded onto SDS-PAGE gels, separated by electrophoresis, andtransferred onto pieces of polyvinylidene fluoride (PVDF) membrane.

The PVDF membranes were analyzed by Western blot. Briefly, the membraneswere washed, blocked, and reacted with rabbit anti-I-κBα,anti-phosphorylated p44/42 mitogen-activated protein kinases (MAPKs),anti-phosphorylated p38 MAPK, or anti-phosphorylated c-Jun N-terminalkinase (JNK) MAPK antibodies in a cold room overnight. After removal ofunbound antibodies by washes, the PVDF membranes were reacted withhorseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody,washed, developed with an Amersham enhanced luminol-basedchemiluminescent (ECL™) kit, and autoradiographed. The PVDF membraneswere then stripped and re-probed with anti-glyceraldehyde-3-phosphatedehydrogenase (GAPDH), anti-p44/42, anti-p38, and anti-JNK antibodies,respectively.

The results are summarized in FIG. 11. As shown in FIG. 11, HMGN1 (SEQID NO: 1) treatment of mouse DCs decreased the level of I-κBα by the 60minute time point, indicating the activation of nuclear factorkappa-light-chain-enhancer of activated B cells (NF-κB) in DCs. Similarband intensity for the three lanes confirms that a similar amount oftotal DC lysate proteins was loaded into each lane.

For the MAPKs, HMGN1 (SEQ ID NO: 1) treatment caused phosphorylation ofthree classes of MAPKs in a time-dependent manner (as evidenced by theintensified bands of phosphorylated p44/42, phosphorylated p38, andphosphorylated JNK), indicating the activation of three classes ofMAPKs. The bands of p44/42, p38, and JNK were similar between 0, 20, and60 minute time points, indicating that HMGN1 (SEQ ID NO: 1) treatmentdid not change the level of unactivated MAPKs.

These results support the use of HMGN1 (SEQ ID NO: 1) to activatedendritic cells and enhance an immune response.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. A method of enhancing an antigen-specificimmune response in a host comprising administering to the host: (i) anantigen and (ii) a polypeptide comprising Nsbp1 (SEQ ID NO: 5), in anamount effective to enhance an antigen-specific immune response.
 2. Amethod of enhancing the activation or recruitment of dendritic cells ina host comprising administering to the host: (i) an antigen and (ii) apolypeptide comprising Nsbp1 (SEQ ID NO: 5), in an amount effective toenhance the activation and recruitment of dendritic cells in the host.3. A method of shifting the Th-1/Th-2 balance of an immune response of ahost towards a Th-1 type immune response comprising administering to thehost (i) an antigen or nucleic acid encoding same, and (ii) apolypeptide comprising Nsbp1 (SEQ ID NO: 5), in an amount effective toshift the Th-1/Th-2 balance of an immune response towards a Th-1 typeimmune response.
 4. The method of claim 1, wherein the method comprisesadministering two or more different antigens to the host.
 5. The methodof claim 1, wherein the antigen is a tumor antigen.
 6. The method ofclaim 5, wherein the tumor antigen is a melanoma antigen.
 7. The methodof claim 1, wherein the method comprises administering a nucleic acidencoding the antigen.
 8. The method of claim 1, wherein the host is ahuman.
 9. The method of claim 2, wherein the antigen is a tumor antigen.10. The method of claim 3, wherein the antigen is a tumor antigen. 11.The method of claim 9, wherein the tumor antigen is a melanoma antigen.12. The method of claim 10, wherein the tumor antigen is a melanomaantigen.
 13. The method of claim 2, wherein the method comprisesadministering two or more different antigens to the host.